Process for the fractionation of polymers

ABSTRACT

A process for the purification or fractionation of aqueous soluble polymers using an aqueous two-phase system is described. The concentrations of the polymer to be fractionated and of an aqueous soluble salt, and the temperature of the aqueous fractionation medium are adjusted so that two phases form, the lower molecular weight polymer molecules partition into the high salt concentration phase, and the higher molecular weight polymer molecules partition into the low salt concentration phase. The resulting high molecular weight polymers are characterized by a higher average molecular weight and a narrower molecular weight distribution and decreased unsaturation than the unfractionated polymers. After being subjected to the fractionation process, polyol polymers that form hydrogels in aqueous solution exhibited higher viscosities and a liquid to gel transition over a narrower temperature range than the unfractionated polyol polymers.

[0001] This invention claims the priority of provisional application No.60/225,917 filed on Aug. 17, 2001.

BACKGROUND

[0002] 1. Field of Invention

[0003] This invention relates to a process for the fractionation ofpolymers that are aqueous soluble, and do not contain functional groupscapable of carrying a charge at neutral pH.

[0004] 2. Description of Prior Art

[0005] Many methods have been described for separating polymers ofsimilar composition and structure. See for example U.S. Pat. Nos.5,028,336, 5,116,508, 5,523,492, 5,567,859, 5,696,298, 5,800,711 andEuropean Patent Application WO 92/16484, incorporated herein byreference. Also, various procedures have been described to fractionateproteins and peptides, but most include precipitation using ammoniumsulfate (Englard and Seifter, 1990). This method relies on the fact thatproteins, in an aqueous solution, maintain a tertiary structure based ontheir amino acid composition and various bonds within the molecule. Thetertiary structure generally allows the hydrophobic substituents to besequestered inside the molecule and the hydrophylic components to be onthe surface, and in contact with the aqueous environment. Changes in theionic strength of the aqueous solution cause unfolding of the molecule,and with exposure of the hydrophobic substituents to the aqueousenvironment, the solubility of the protein decreases and itprecipitates. By carefully adjusting pH, ionic strength, and sometimestemperature, it is frequently possible to separate proteins with similaramino acid sequences (Englard and Seifter, 1990, King, 1972, Tarli andLi, 1974). Clearly, this method is useful only for proteins and otherpolymers composed of substituents that vary considerably in polarity,and therefore aqueous solubility. This is not the case for manysynthetic polymers.

[0006] A method to separate water soluble organic electrolytes in anaqueous medium from other water soluble hydrocarbons has been disclosed(Bartels and Reale, U.S. Pat. No. 5,028,336). The pH of the aqueousmedium is adjusted so that most of the organic electrolytes are charged.The aqueous medium is then passed through a filtration membrane whichcarries the same charge. The organic electrolytes are repelled by thecharge on the membrane, and therefore do not cross. Water and unchargedorganic molecules pass through the membrane and are thus separated fromthe organic electrolytes. This method is limited to organic moleculessuch as carboxylic acids which contain functional groups capable ofcarrying a charge at some pH.

[0007] Methods relying on supercritical fluid extraction have beendeveloped to separate high molecular weight compounds, includingpolymers, from complex mixtures in aqueous solution (See for exampleKumar and Hedrick, U.S. Pat. No. 5,116,508). This method requires amobile phase of highly compressed gas, such as CO₂, at or above itscritical temperature and pressure, to be pumped through the aqueoussolution. The composition of the mobile phase can be modified to enhanceextraction of the desired analyte. Such modifications include using amixture of gases as the mobile phase, or adding a modifying chemical tothe supercritical fluid. Such methods can be conducted on a commercialscale, and used to separate uncharged polymers including polyols.Nevertheless, to be effective, the compressed gases often must bemaintained at high temperatures and pressures, which requires complex,well controlled equipment. This frequently makes supercritical fluidextraction an expensive process and limits its commercial applications.

[0008] Synthetic polyols such as poly(ethylene glycol) andpolyoxyalkylene block copolymers have been used in various medical andpharmaceutical applications including treatment of sickle cell disease,reduction of blood viscosity, treatment of tissue ischemia, treatment oftissue following electrical injury, and drug delivery (Emanuele U.S.Pat. No. 5,691,387, Lee, R. C., U.S. Pat. No. 5,605,687, Reeve, L. E.,1997). These linear polymers are generally synthesized by repeatedsequential reactions that add monomeric subunits to each end of thepolymeric chain. Since subunits may add to either or both ends ofindividual chains at variable rates, the end product is a mixture ofmolecules varying in molecular weight.

[0009] The poly(ethylene glycol)s are composed entirely of ethyleneoxide residues linked by ether linkages and vary considerably inmolecular weight. These synthetic polymers have been used extensively indrug delivery to solubilize pharmaceutically active compounds. Recently,they have been used to derivatize proteins, peptides and small moleculesto prolong half-life and enhance delivery within the body ( ). They havealso been derivatized and used as cross-linking components in medicaldevices. For optimal safety and efficacy in medical applications, theserecent uses require polymers of uniform molecular weight having minimalcontamination with reaction byproducts.

[0010] The poloxamers are polyoxyalkylene block copolymers composed oftwo polyexyethylene blocks separated by a polyoxypropylene center block.The commercially available product contains a mixture of polyoxyethylenehomopolymer, and polyoxyethylene/polyoxypropylene diblock polymers inaddition to poloxamer molecules of varying molecular weights. Thesefactors cause the polymer product to have a broad molecular weightrange, reflected in a high polydispersity index. The mono- and diblockpolymers are generally of a lower molecular weight than the average forthe polymer product and contain some unsaturation. When commerciallyavailable poloxamers (purchased from BASF Corp.) were analyzed by gelpermeation chromatography, a bimodal molecular weight distribution wasobserved (Reeve, L. E., 1997). The mono- and diblock contaminants,including the unsaturated species, partitioned into the lower molecularweight fraction.

[0011] European Patent Application WO 92/16484 discloses the use of gelpermeation chromatography to isolate a fraction of poloxamer 188 thatexhibits beneficial biological effects, without causing potentiallydeleterious side effects. The copolymer thus obtained had apolydispersity of 1.07 or less, and was substantially saturated. Thepotentially harmful side effects were shown to be associated with thelow molecular weight, unsaturated portion of the polymer, while themedically beneficial effects resided in the uniform higher molecularweight material. Other similarly improved copolymers were obtained bypurifying either the polyoxypropylene center block during synthesis ofthe copolymer, or the copolymer product itself (Emanuele U.S. Pat. No.5,523,492, Emanuele U.S. Pat. No. 5,696,298). Although an effectivemeans of purification, gel permeation chromatography is impractical forthe preparation of large quantities of the fractionated polyoxyalkyleneblock copolymer.

[0012] A super critical fluid extraction technique has been used tofractionate a polyoxyalkylene block copolymer as disclosed in U.S. Pat.No. 5,567,859. A purified fraction was obtained, which was composed of afairly uniform polyoxyalkylene block copolymer having a polydispersityof less than 1.17. According to this method, the lower molecular weightfraction was removed in a stream of CO₂ maintained at a pressure of 2200pounds per square inch (psi) and a temperature of 40° C. As isfrequently the case, this super critical fluid extraction methodrequired equipment that can control temperature and accommodatecompressed CO₂ at high pressure. Clearly, these requirements add expenseto the procedure and limit its commercial value.

[0013] U.S. Pat. No. 5,800,711 discloses a process for the fractionationof polyoxyalkylene block copolymers by the batchwise removal of lowmolecular weight species using a salt extraction and liquid phaseseparation technique. Poloxamer 407 and poloxamer 188 were fractionatedby this method. In each case, a copolymer fraction was obtained whichhad a higher average molecular weight and a lower polydispersity indexas compared to the starting material. However, the changes inpolydispersity index were modest and analysis by gel permeationchromatography indicated that some low molecular weight materialremained. The viscosity of aqueous solutions of the fractionatedpolymers was significantly greater than the viscosity of thecommercially available polymers at temperatures between 10° C. and 37°C., an important property for some medical and drug deliveryapplications. Nevertheless, some of the low molecular weightcontaminants of these polymers are thought to cause deleterious sideeffects when used inside the body, making it especially important thatthey be removed in the fractionation process. As a consequence,polyoxyalkylene block copolymers fractionated by this process are notappropriate for some medical uses.

[0014] Aqueous two phase systems have been used extensively toconcentrate or isolate polymers, other large molecules, and evenparticles from complex mixtures (Hatti-Kaul,R., 2000). Such systemsgenerally avoid the use of organic solvents, and extremes of pH ortemperature, and, because of their mild conditions, have been shown tobe especially useful to isolate amino acids, peptides, proteins, plasmamembranes including membrane vesicles, and viruses. These systems arecomposed of either hydrophilic polymer pairs or a polymer and a saltthat are incompatible in aqueous solution and form two phases inequilibrium with each other. Separations can be carried out using eitherbatch procedures or counter-current distribution. Although widely usedfor the isolation and purification of biomaterials, aqueous two-phasesystems have been used much less extensively for the isolation orfractionation of synthetic polymers.

SUMMARY

[0015] The present invention is a method for the fractionation ofwater-soluble synthetic polymers by physically separating the lowermolecular weight polymer molecules, byproducts, and contaminants fromthe desired polymer by using an aqueous two-phase system.

OBJECTIVES AND ADVANTAGES

[0016] The present invention provides a process for the fractionation ofaqueous soluble polymers comprising dissolving a known amount of thepolymer in water, adding a soluble extraction salt to the polymersolution, maintaining the solution at a constant optimal temperature fora period of time adequate for two distinct phases to appear, andphysically separating the phases. Additionally, the phase containing thepolymer fraction of the preferred molecular weight may be diluted to theoriginal volume with water, extraction salt added to achieve theoriginal concentration, and the separation process repeated as neededuntil a polymer having a narrower molecular weight distribution than thestarting material and optimal physical characteristics can be recovered.

[0017] It is an object of the present invention to provide a method thatseparates lower molecular weight polyol polymer molecules from those ofvery similar chemical composition but higher molecular weight therebynarrowing the molecular weight distribution.

[0018] It is another object of the present invention to provide a methodto remove reaction byproducts from the polyol polymer.

[0019] Another object of the present invention is to provide an improvedmethod which produces a polyol polymer with improved chemical andphysical characteristics including decreased polydispersity, decreasedunsaturation, higher viscosity in aqueous solution, and hence increasedgel strength, and a well defined transition temperature for thosepolyols that form gels in solution.

BRIEF DESCRIPTION OF THE INVENTION

[0020]FIG. 1 is a chromatogram of poly(ethylene glycol) showing themolecular weight distribution of the polymer before and afterfractionation using the method of the present invention. Thechromatogram was obtained by gel permeation chromatography using two 7.8mm×25 cm columns packed with styrene/divinyl benzene copolymer gel (100μm pore size) eluted with a solvent composed of 98:2tetrahydrofuran/triethylamine and a refractive index detector.

[0021]FIG. 2a is a chromatogram of poloxamer 407 showing the molecularweight distribution of the polymer before and after fractionation usingthe method of the present invention. The same chromatographic systemdescribed for FIG. 1 was used.

[0022]FIG. 2b is a comparison of the viscosities of a 25% solution ofcommercially available poloxamer 407 and fractionated poloxamer 407obtained from the method of the present invention. Viscosities weremeasured over a temperature range of about 0° C. to about 45° C.

[0023]FIG. 3a is a chromatogram of poloxamer 188 showing the molecularweight distribution of the polymer before and after fractionation usingthe method of the present invention. The chromatographic systemdescribed in FIG. 1 was used.

[0024]FIG. 3b is a comparison of the viscosities of a 35% aqueoussolution of commercially available poloxamer 188 and fractionatedpoloxamer 188 obtained from the method of the present invention.

[0025]FIG. 4a is a chromatogram of poloxamine 1307 showing the molecularweight distribution of the polymer before and after fractionation usingthe method of the present invention. The chromatographic systemdescribed for FIG. 1 was used.

[0026]FIG. 4b is a comparison of the viscosities 25% aqueous solution ofcommercially available poloxamine 1307 and fractionated poloxamine 1307obtained from the method of the present invention. The viscosities ofeach solution were measured over a temperature range of about 0° C. toabout 50° C.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Although molecular weight averages are informative when comparingpolymers, it is also useful to know the molecular weight distribution ofeach polymer. The polydispersity index of a polymer is a universallyaccepted measure of the breadth of the molecular weight distribution andis defined as follows:

[0028] Number Average Molecular Weight (M_(n)):

M _(n)=Sum of (W _(i)) for all/Values/Sum of (W _(i) M _(i))

[0029] Weight Average Molecular Weight (M_(w)):

M _(w)=Sum of [(W _(i))(M _(i))] for all/Values/Sum of (W _(i)) forall/Values

[0030] Polydispersity Index (D):

(D)=M _(w) /M _(n)

[0031] where W_(i) is the mass of the i-th polymer species and M_(i) isthe molecular weight of the i-th species. A low polydispersity value, D,indicates a narrow molecular weight distribution. In a monodispersepopulation where all molecules are identical, M_(w) would be equal toM_(n) and the polydispersity index would be equal to 1.0. Typicalpolymer preparations have polydispersity index values between 1.5 and 5,but some may be much higher.

[0032] A variety of procedures is available for determining molecularweight including gel permeation chromatography or other chromatographictechniques, viscosity related measurements, light scattering, osmoticpressure, ultra centrifugation and chemical methods involving end groupanalysis. For most polymers molecular weight distribution parametersincluding M_(w), M_(n), and D, are conveniently measured using gelpermeation chromatography.

[0033] The viscosity of a fluid is a measure of friction that resultswhen one layer of the fluid to moves in relation to another layer inresponse to a shearing force. The terms shear stress (τ) and rate ofshear (γ) are used to indicate the applied force and the response of thefluid (Rodriguez, 1989). Shear viscosity is defined as:

[0034] τ=shear stress=f/A

[0035] γ=rate of shear=u/y

[0036] η=shear viscosity=τ/γ

[0037] where f/A is the force per unit of area required to maintain aconstant velocity gradient, u/y. Viscosity is expressed asPascal(seconds), or centipoise (cps), where 1000 cps equals 1Pascal(second).

[0038] The process of the present invention separates lower molecularweight polymer molecules and byproducts from high molecular weightpolyol polymer molecules. The process consists of forming an aqueoustwo-phase system composed of the polymer and an appropriate salt inwater. In such a system, a soluble salt can be added to a single phasepolymer-water system to induce phase separation to yield a high salt,low polymer bottom phase, and a low salt, high polymer upper phase.Under carefully selected conditions lower molecular weight polymerspartition preferentially into the high salt, low polymer phase. Polymersthat can be fractionated using this process include polyethers, glycolssuch as poly(ethylene glycol) and poly(ethylene oxide)s, polyoxyalkyleneblock copolymers such as poloxamers, poloxamines, andpolyoxypropylene/polyoxybutylene copolymers, and other polyols such aspolyvinyl alcohol. The average molecular weight of these polymers mayrange from about 800 to greater than 100,000 daltons.

[0039] The poloxamers are a series of block copolymers having thegeneral structure:

HO(C₂H₄O)_(a)(C₃H₆O)_(b)(C₂H₄O)_(a)H

[0040] The average molecular weights of the poloxamers range from about1,000 to greater than 16,000 daltons. Because the ploxamers are productsof a sequential series of reactions, the molecular weights of theindividual poloxamer molecules form a statistical distribution about theaverage molecular weight. In addition, commercially available poloxamerscontain substantial amounts of poly(oxyethylene) homopolymer andpoly(oxyethylene)/poly(oxypropylene diblock polymers. The relativeamounts of these byproducts increase as the molecular weights of thecomponent blocks of the poloxamer increase.

[0041] Depending upon the manufacturer, these byproducts may constitutefrom about 15 to about 50% of the total mass of the polymer. The processof the present invention exploits the differences in size and polarity,and therefore solubility, among the poloxamer molecules, thepoly(oxyethylene) homopolymer and thepoly(oxyethylene)/poly(oxypropylene) diblock byproducts. The polarfraction of the poloxamer, which generally includes the lower molecularweight fraction and the byproducts, is removed allowing the highermolecular weight fraction of poloxamer to be recovered. The largermolecular weight poloxamer recovered by this method has physicalcharacteristics substantially different from the starting material orcommercially available poloxamer including a higher average molecularweight, lower polydispersity and a higher viscosity in aqueous solution.

[0042] The poloxamines are tetra-functional block copolymers synthesizedby the sequential addition of first, propylene oxide, and then ethyleneoxide to the tertiary nitrogens of ethylenediamine. The poloxamines havethe following general structure:

[0043] Like the poloxamers, the poloxamines are composed of moleculesthat vary considerably in molecular weight. When subjected to gelpermeation chromatography, commercially available poloxamine (Tetronic®1307 purchased from BASF Corp., Mount Olive, N.J.) eluted as threeseparate peaks (FIG. 4a). The process of the present invention removedmuch of the lower molecular weight material, producing a polymer with aslightly higher average molecular weight and more uniform size comparedto the starting material. In addition, the viscosities of aqueoussolutions of the fractionated polymer were considerably higher thanthose of the commercially available material.

[0044] Because they are composed of hydrophobic poly(oxypropylene)blocks and hydrophilic poly(oxyethylene) blocks, both poloxamers andpoloxamines form micelles in aqueous solutions. If the concentration ofthe polymer is sufficient, the micelles aggregate in a characteristic,temperature dependent fashion, and cause the solution to become ahydrogel. Such hydrogels have been used in various medical applicationsincluding site specific drug delivery and manipulation of tissue duringand after surgery (Reeve, 1997). Of particular interest are poloxamer407 and poloxamine 1307 because, for these polymers, the transition fromliquid to gel takes place below 37° C. Therefore, in medicalapplications, a formulation containing these polymers can be applied tothe human body as a liquid at or below room temperature that will coatand adhere to tissues, but will rapidly form a gel as it equilibrates tobody temperature, and remain where it is placed. However, for bothcommercially available poloxamer 407 and poloxamine 1307, gelationoccurs over a fairly broad temperature range of ten to twenty degreesCelsius. For fractionated polymers recovered from the process of thepresent invention, in contrast, the transition from liquid to geloccurred in a much narrower, well defined temperature range ofapproximately two to five degrees Celsius. In addition, the viscositiesof gels of various concentrations of either fractionated polymer werehigher above 30° C. These two characteristics of the fractionatedpolymers, rapid transition from liquid to gel over a narrow temperaturerange, and higher viscosity at body temperature, provide an improved gelfor medical applications. Lower concentrations of the fractionatedpolymer can be used to provide a reliable formulation that will rapidlybecome a gel at a well defined temperature, but with reduced exposure tothe polymer for the patient.

[0045] The commercially available poly(ethylene glycol)s andpoly(ethylene oxide)s are polymers composed of ethylene oxide monomersheld together by ether linkages. The poly(ethylene glycols) vary fromseveral hundred to approximately 20,000 average molecular weight. Themolecular weights of the poly(ethylene oxide)s range from above 20,000to several million. Like the polyoxyalkylenes, these polymers aresynthesized by the repeated addition of monomers to the ends of thegrowing polymer chain, and therefore, are composed of molecules varyingin molecular weight, and include some relatively low molecular weightmaterial (FIG. 1). The process of the present invention was found toremove the lower molecular weight material yielding a more uniformproduct. The poly(ethylene glycol)s have been used in aqueous two phasesystems as the “incompatible” polymer to concentrate and isolatebiopolymers. However, the poly(ethylene glycol)s, themselves have notbeen fractionated or purified by such methods.

[0046] The process of the present invention comprises the followingsteps:

[0047] 1. A known amount of the polyol polymer to be fractionated isdissolved in water at an appropriate concentration.

[0048] 2. The mixture is equilibrated to about 0 to 5° C., then asoluble extraction salt is added slowly with vigorous mixing until thesolution becomes opaque. Soluble salts may include salts of sulfate,phosphate and citrate. The preferred salts are sulfates, such asammonium sulfate ((NH₄)₂SO₄). Dextran and starch may also be used.

[0049] 3. The solution is allowed to equilibrate at between 0 and 5° C.until two distinct phases, upper and lower, appear (usually betweenabout 2 and about 8 hours). Centrifugation may be used to expedite phaseseparation.

[0050] 4. The lower layer is removed. The upper layer is diluted to itsoriginal volume by the addition of deionized water.

[0051] 5. Steps 2,3, and 4 are repeated from about 2 to about 5 timesdepending upon the polyol polymer used as the starting material, thecontaminating byproducts and the degree of fractionation required.

[0052] 6. After the final extraction, the upper layer containing thefractionate of the polyol polymer may the isolated and/or concentratedby extraction into dichloromethane, chloroform or any other suitableorganic solvent or solvent mixture, or by dialysis. If organicextraction is used, the extract may be dried using a suitable agent suchas anhydrous sodium sulfate.

[0053] 7. Residual solvent(s) can be removed by vacuum orlyophilization.

[0054] 8. The higher molecular weight fraction of the polyol polymer isthe dried residue obtained after removal of the solvent.

[0055] In the case of the poloxamers and the poloxamines, thefractionated polymer has a reduced polydispersity index, reducedunsaturation and increased viscosity in aqueous solution compared to thestarting material which allows for better functionality for variousmedical and pharmaceutical applications. In the case of poly(ethyleneglycol), the fractionated polymer has a higher average molecular weightand a narrower molecular weight distribution than the starting materialas indicated by a lower polydispersity index.

[0056] The following examples illustrate the fractionation process.

EXAMPLE 1

[0057] Poly(ethylene glycol) (lot number 15628B0) (mwt 10,000) purchasedfrom Aldrich Chemical Corp., Milwaukee, Wis., was weighed and 0.45 gramswere dissolved in 15 grams of deionized water. The solution wasmaintained at 1.5° C. and 2.51 g of (NH₄)₂SO₄ were added slowly untilthe solution turned opaque. Solution was maintained at 2° C., and aftertwo phases formed, the lower phase was removed. Deionized water (13.2 g)was added and the solution was equilibrated at approximately 1° C. Next,(NH₄)₂SO₄ (2.3 g) was added with stirring until the solution turnedopaque. The solution was maintained at approximately 2° C. until twophases separated. The lower phase was removed, 12.9 g of deionized waterwas added, the solution was chilled to 1.3° C., and 2.4 g (NH₄)₂SO₄ wereadded. The solution was maintained at approximately 2° C. until twophases separated. The upper phase was isolated, washed into a separatoryfunnel with 30 ml of deionized water and extracted three times with 10ml dichloromethane. The dichloromethane extracts were combined, and thesolvent was removed under vacuum at 20° C. The fractionated polyethyleneglycol (0.11 g) was recovered, and found to have an average molecularweight of 11,168 compared to 10,588 for the commercially availablepoly(ethylene glycol) starting material and a polydispersity index of1.028 compared to 1.059 for the starting material.

EXAMPLE 2

[0058] Poloxamer 407 (lot number WPHT-543B), purchased from BASFCorporation, Mount Olive, N.J., was weighed and 486.0 g were dissolvedin 15,733 g of deionized water. The solution was maintained at 0.1° C.and 2335.1 g of (NH₄)₂SO₄ were added. The solution was equilibrated at2° C. and after two distinct phases formed, the lower phase wasdiscarded, and the upper phase (2060 g) was collected and weighed.Deionized water (14159 g) was added and the solution was equilibrated to2° C. Next, 2171.6 g of (NH₄)₂SO₄ were added with stirring. After thesalt was dissolved, the solution was maintained at approximately 2° C.until two phases formed. The upper phase (3340 g) was isolated anddiluted with 12879 g of deionized water. The solution was chilled toabout 2.2° C. and 2062 g of (NH₄)₂SO₄ were added. The phases wereallowed to separate as above. The upper phase was isolated and extractedwith 4 liters of dichloromethane. Two phases were allowed to formovernight. The organic (lower) phase was isolated and approximately 2 kgof sodium sulfate (Na₂SO₄) were added to it to remove the remainingwater. The dichloromethane phase was filtered through a PTFE filter(0.45 μm pore size) to remove the undissolved salts. The dichloromethanewas removed under vacuum at approximately 30° C. Final traces ofdichloromethane were removed by drying in an oven overnight at about 30°C. A total of 297.6 g of fractionated poloxamer 407 (lot number00115001) were recovered. The chemical and physical characteristics ofthe fractionated poloxamer 407 are compared to those of the startingmaterial in Table 1. TABLE 1 M_(w)/ Unsaturation Weight % Viscosity,Sample M_(w) M_(n) M_(n) mEq/g oxyethylene centipoise* Poloxamer 40711,996 9,979 1.20 0.048 73.2 275,000 Poloxamer 407, lot 00115001, 13,55112,775 1.06 0.005 69.3 >820,000 fractionated

EXAMPLE 3

[0059] Poloxamer 188 (BASF Corp. Lot # WPMO-568B) was weighed and 4.5 gwere dissolved in 145.5 g of deionized water. The solution was cooled to2° C. and 26.0 g (NH₄)₂SO₄ were added and dissolved with stirring. Thesolution was maintained at approximately 2° C. until the phasesseparated. After two phases formed, the lower phase was discarded, andthe upper phase was diluted with 125.4 g with deionized water. Thesolution was cooled to 0.3° C. and 20.7 g of (NH₄)₂SO₄ were added slowlywith stirring until the solution turned opaque. The solution wasmaintained at 3° C. until two phases formed. After the phases separated,the upper phase was isolated and diluted with 125.4 g with deionizedwater, chilled to −0.4° C. and 21.9 g of (NH₄)₂SO₄ were added to thesolution and dissolved with stirring. The solution turned opaque, andwas then maintained at 3° C. until two clear phases formed. The lowerphase was removed, and the upper phase was diluted with 25 ml deionizedwater. The diluted solution was then extracted three times with 15 mlportions of dichloromethane. The dichloromethane extracts were combinedand washed two times with 25 ml portions of deionized water. The waterextracts were discarded. The dichloromethane extract was first dried byfiltering through anhydrous Na₂SO, and then the solvent was removedunder vacuum at 30° C. The remaining solid material was the fractionatedpoloxamer 188. The fractionated poloxamer 188 was weighed (1.35 g),assigned the lot number 01199001, and its physical properties wereevaluated.

[0060] Both the very high and very low molecular weight material wasremoved from the fractionated poloxamer 188 (FIG. 3a) and thepolydispersity was reduced from1.062 for the unfractionated poloxamer188 to 1.041. The average molecular weight increased from 7,802 to8,212. The viscosity of a 35% w/w aqueous solution of fractionatedpoloxamer 188 began to increase above 35° C., and formed a gel at (andabove) 40° C. In contrast, a 35% solution of commercially availablepoloxamer 188 exhibited an increase in viscosity only above 40° C., andformed a weak gel at approximately 45° C. (FIG. 3b).

EXAMPLE 4

[0061] Poloxamine 1307 (BASF Corp, Mount Olive, N.J., Tetronic 1307® LotNo. WPET-587B) was weighed and 0.45 g were dissolved in 15 g deionizedwater with stirring. The solution was chilled to 1.5° C., and 2.28 g(NH₄)₂SO₄ were slowly added. The solution was maintained at 2° C. untiltwo phases formed. The lower phase was removed, 12.8 g of deionizedwater were added, and the solution was cooled to 0.6° C. Next, 2.0 g(NH₄)₂SO₄ were added slowly with stirring. The solution was thenmaintained at 2° C. without stirring until two phases formed. The lowerphase was removed, 12.7 g of deionized water were added, and thesolution was cooled to 1.40C. (NH₄)₂SO₄ (2.1 g) was added slowly, withstirring until the solution turned opaque. The solution was maintainedat 2° C. until two phases formed.

[0062] The upper phase was isolated and transferred to a separatoryfunnel with the addition of 30 ml of deionized water. The upper phasewas then extracted three times with 10 ml dichloromethane. Thedichloromethane extracts were combined, and the solvent was removedunder vacuum at 20° C. The resulting solid material (0.19 g) was thefractionated poloxamine.

[0063] The average molecular weight of the fractionated polymer was16,217 and the polydispersity was 1.064, compared to an averagemolecular weight of 14,409 and a polydispersity index of 1.316 for thecommercial poloxamine, Tetronic® 1307. A 25% solution of fractionatedpoloxamine 1307 changed from a liquid to a very stiff gel (viscositygreater than 800 kcps) between 20 and 24° C. In contrast the viscosityof a 25% solution of commercially available poloxamine 1307 began toincrease only above 25° C., and formed a non-flowable gel at (and above)30° C. The maximum viscosity was 494 kcps, and occurred at 40° C. (FIG.4b).

[0064] These examples should not be construed as limiting the scope ofthe invention, but as merely providing illustrations of some of thepresently preferred embodiments to this invention.

1-21. (canceled)
 22. A method for the fractionation of polyoxyalkylenepolymers, comprising: a) dissolving up to about 10% by weight ofpolyoxyalkylene polymer in water to form a solution of a knownconcentration of polyoxyalkylene polymer; b) adjusting the temperatureof the solution from step a) to a preselected temperature of betweenabout −5° C. and 30 ° C.; c) adding about 5% to about 25% by weight of asalt to the solution of step b) to provide a known concentration of saltin the solution; and d) allowing the aqueous solution to form a highsalt phase and a low salt phase at the preselected temperature; whereinthe concentration of the polyoxyalkylene polymer, the concentration ofthe salt, and the preselected temperature are chosen so that the lowermolecular weight polyoxyalkylene polymer molecules partition into thehigh salt concentration phase and the higher molecular weightpolyoxyalkylene polymer molecules partition into the low saltconcentration phase of the aqueous solution.
 23. The method of claim 22,wherein the salt is a sulfate, citrate, or phosphate.
 24. The method ofclaim 22, wherein the salt is a sulfate.
 24. The method of claim 22,wherein the salt is a citrate.
 25. The method of claim 22, wherein thesalt is a phosphate.
 26. The method of claim 22, wherein the salt isammonium sulfate or sodium sulfate.
 27. The method of claim 22, whereinthe salt is ammonium sulfate.
 28. The method of claim 22, wherein thesalt is sodium sulfate.
 29. The method of any of claims 22-28, whereinthe polyoxyalkylene polymer has an average molecular weight of about3,000 daltons to about 100,000 daltons.
 30. The method of any of claims22-28, wherein the polyoxyalkylene polymer is selected from the groupconsisting of poly(alkylene glycol), poly(alkylene oxide), andpolyoxyalkylene block copolymers.
 31. The method of any of claims 22-28,wherein the polyoxyalkylene polymer is a polyoxyakylene block copolymer.32. The method of any of claims 22-28, wherein the polyoxyalkylenepolymer is selected from the group consisting of poly(ethylene glycol),poly(ethylene oxide), poloxamers, and poloxamines.
 33. The method of anyof claims 22-28, wherein the polyoxyalkylene polymer is selected fromthe group consisting of poly(ethylene glycol), poloxamers, andpoloxamines.
 34. The method of any of claims 22-28, wherein thepolyoxyalkylene polymer is poly(ethylene glycol).
 35. The method of anyof claims 22-28, wherein the polyoxyalkylene polymer is a poloxamer. 36.The method of any of claims 22-28, wherein the polyoxyalkylene polymeris a poloxamine.